It is generally accepted that cells cultured in a 3D configuration are physiological more relevant than cells in classical monolayer cultures (see e.g. Yamada and Cukiermann, Cell, 2007; Pamploni et al. Nature Reviews Molecular Cell Biology, 2007). Coaxing cells into the third dimension is the quintessential design problem. Current technologies are mostly based either on the use of scaffold materials or stacking of monolayers to shape the cells. However, despite the biological benefit, current state-of-the-art technologies are not laboratory routine or used on an industrial scale for applications such as drug discovery or toxicity assays given that the cell culture process is more complex, time-consuming and requires additional biomaterials. The re-aggregation of cells is an alternative approach to coax cells into the third dimension. But current re-aggregation technologies have been proven mostly with neoplastic cell lines and lack controlled co-culture possibilities. The hanging drop (HD-) technology has shown to be a universal method to enable 3D cell culture with neoplastic as well as primary cells (see Kelm and Fussenegger, 2004, Trends in Biotechnology Vol. 22, No. 4: 195-202). Drops of cell culture medium with suspended cells are placed onto a cell culture surface and the plate is inverted. As there is no substrate available on which the cells can adhere, they accumulate at the bottom of the drop and form a microtissue.
Cultivation of cells in drops that are hanging at a surface is well known to the person of skill in the art. Form DE 103 62 002 B4, for example, the usual way of depositing drops of a cell suspension in a nutrient medium with a pipette on the inner surface of a Petri dish cover is known. The Petri dish cover then has to be inverted and placed on an appropriate Petri dish base plate. In the so closed Petri dish, the drops hang from the cover surface. The Petri dish often contains wet filter paper for providing the hanging drops with a humid atmosphere that prevents the hanging drops from drying. One of the most critical steps of this conventional hanging drop technique is inverting the plate to which the drops are attached; thus, this crucial step very often has to be carried out manually by an experienced scientist.
From WO 03/078700 A1, the application of the hanging drop technique is known for culturing stem cells and for the production of protein crystals. The advantages of the hanging drop technology comprise the fact that the substances under investigation are completely surrounded with the nutrient medium that provides all factors needed, such as ions, differentiation factors, toxic substances etc. In addition, aggregation of cells (e.g. stem cells) is promoted in that the cells sink to the apex of the drop where they meet and form a cluster (e.g. embryonic bodies) without having touched a solid surface. The surface tension of the drop prevents the cells as well as the cell aggregates from penetrating the droplet surface. However, the drops applied with a pipette may comprise only a small volume as the drops may move on the surface during inverting the surface for providing the correct position to establish hanging drops. In order to provide larger drops of equal dimension and thus enabling identical cultivation or reaction environments, sharp-edged relief structures that limit a drop contact area on a particular surface are proposed.
More recently (see e.g. Kelm et al. 2004 or Khademhosseini et al. 2006, PNAS Vol. 103, No. 8: 2480-2487), cell culturing in hanging drops has been called microscale tissue engineering using gravity-enforced cell assembly. Whereby Khademhosseini et al. seem to favor microscale tissue engineering using template-based cell assembly in polyethylene glycol (PEG) microwells; Kelm and Fussenegger apply the hanging drop technique in wells of a multiwell or Terasaki plate.
All these documents report the necessity of inverting the substrate to which the drops adhere in order to correctly provide them as hanging drops. After being inverted, the substrates are reported to lay horizontal or to include an angle of at most 90° with the horizontal direction (see WO 03/078700 A1). Such inverting is difficult to handle manually and even more difficult to carry out by a robot. Thus, the required manual inversion of the plate impedes mass production and automation compatibility.